1. Field of the Invention
The present invention relates to methods for diagnosing plant diseases.
2. Prior Art
The classical and accepted methods for diagnosing bacterial and fungal diseases in plants involves isolating the microbe in pure culture from a diseased plant, inoculating test plants, and reisolating the microbe from the test plant after observing the same disease symptoms of those originally found. This procedure is tedious, time-consuming and requires a high level of experience with the plant and with the suspected microorganisms. Moreover, the microbes; because of their plant pathogenic growth preferences, very often grow slowly or not at all in pure culture.
The reliable diagnosis of plant diseases, therefore, requires sophisticated laboratories, equipment and highly-trained personnel. In practice and in the field, amateur and small-scale agricultural growers do not have ready access to the experienced personnel and/or laboratories necessary for these diagnoses. The time, effort and costs involved are not justified by the limited utility of the information to the small-scale grower. Even on a large-scale, decisions as to frequently costly intervention measures must be made quickly in order to save the plants infected by the suspected diseases. The time frame required for a confirmed diagnosis according to the classical method is unduly lengthy. Therefore, expediency requires that such decisions be made on the basis of prior experience with the disease and the specific infected plant type. These decisions may prove to be erroneous in the worst cases, thereby resulting in the loss of valuable plants and/or the utilization of improper methods of treatment.
It has been proposed to utilize "recombinant DNA(rDNA) probes" containing cloned fragments of DNA specific for selected viral microbes to identify viral diseases in plants. See U.S. Pat. No. 4,480,040; Gould et al, Journal of Virological Methods, Vol. 2, pp. 287-292 (1981); Eden et al, Journal of Bacteriology, August 1974, pp. 547-553; Allen et al, Ann. Appl. Biol., Vol. 98, pp. 451-461 (1981); Palukaitis et al, Ann. Appl. Biol., Vol. 98, pp. 439-449 (1981).
The procedure described in U.S. Pat. No. 4,480,040 is typical of the prior art DNA probe techniques for diagnosing viral and viroid diseases in plants. Generally, these techniques require a time-consuming and expensive preparation of a purified or partially purified plant extract to conduct the diagnosis. The need for an extraction or partial purification step in order to successfully detect diseases in plants necessarily lengthens the time required and increases the cost for successfully diagnosing plant viral diseases.
The distinctions between bacterial and fungal diseases on the one hand and viral diseases on the other is well known to those skilled in the art. The detection of viruses and viroids normally requires the use of biochemical diagnostic techniques such as serology, protein electrophoresis and/or DNA hybridizations. The diagnosis of bacterial and fungal diseases is relatively simpler in that they are readily identifiable by simple microbial tests after culturing. Viral diseases are not amenable to this type of diagnosis because they cannot be cultured, and the more sophisticated laboratory tests are necessary. However, even classical methods of diagnosing bacterial and fungal diseases are expensive, lengthy and time-consuming as noted above.
The application of DNA hybridization technology to plant disease detection and diagnosis was developed by necessity as the only rapid means available for the detection of viroids in plants [Owens, et al (1981), Science 213:670-672]. Since viroids consist of replicating, unencapsulated RNA, direct hybridization of complementary DNA (cDNA) probes to plant cell-sap concentrates was a workable idea. Since unencapsulated RNA or DNA is an important stage of the life cycle of viruses, the same technique could be directly applied to viral plant pathogens. However, since bacterial and fungal DNA is contained within the protective cell walls of these organisms (as opposed to viruses and viroids), the DNA thereof is obviously not available for hybridization without removal from within the cells using an extraction procedure.
Although in situ lysis and hybridization procedures have been developed for bacteria and fungi [Grunstein et al (1975), Proc. Natl. Acad. Sci., USA 72:3961-3965, and Stohl et al (1983), Anal. Biochem. 134:82-85], all such techniques rely upon macroscopically visible bacterial or fungal colonies grown on culture media. Bacterial and fungal disease organisms which produce leaf lesions in plants are generally not known to achieve sufficiently high population levels within the leaf to allow ready detection with current DNA probe technology. Those skilled in the art would conclude that it would first be necessary to culture the microbes outside of the leaf to achieve a sufficiently high population for detection.
It has been proposed to use rDNA probes to detect bacterial pathogens of human in excreta or physiological fluid, without extraction or purification [U.S. Pat. No. 4,358,535, Dallas et al (1979). Proceedings of the 13th Joint Conference on Cholera, U.S. Department of Health, Education and Welfare publication No. NIH 78-1590, p. 71-80, NIH, Bethesda, Md., and Dallas et al (1979), in K. N. Timmis and A. Puhler (ed.), Plasmids of medical, environmental, and commercial importance, Elsevier/North Holland Publishing Co., Amsterdam, p. 113-122].
It is an object of the present invention to provide a rDNA probe technique for detecting or diagnosing bacterial and fungal diseases in plants which produce leaf lesions, said procedure being efficient and inexpensive and capable of being effectuated by relatively untrained personnel and without the requirement for sophisticated equipment. The invention is unique in that no partial purification, concentration or extraction procedure or apparatus designed for these purposes need be employed.